Lyophilized aryl phosphate monoesters and process therefor

ABSTRACT

Lyophilized aryl phosphate monoesters having a pH of substantially 1 to 4 upon reconstitution with the amount of water removed during lyophilization; are prepared by lyophilizing an aqueous solution of the aryl phosphate monoesters having a pH of substantially 1 to 4. Diagnostic compositions for the determination of phosphates in which the lyophilized aryl phosphate monoesters are employed.

United States Patent 91 Hammer [54] LYOPHILIZED ARYL PHOSPHATE MONOESTERS AND PROCESS THEREFOR [75] Inventor: Frank E. Hammer, Chicago, Ill. [73] Assignee: G. D. Searle&C0.,Chicag0, Ill. 22 Filed: Sept. 8, 1969 21] Appl.No.: 856,205

1 1 Mar. 27, 1973 OTHER PUBLICATIONS Bessey et-al., Jou. of Biological Chem. Vol. 196, pp. 175-178, (1952).

Primary Examiner-Lewis Gotts Assistant Examiner-Anton H. Sutto Attorney-John M. Brown, John J. Kolano, Elliot N.

Schubert, Sybil Meloy, .Walter C. Ramm and Helmu A. Wegner [57] ABSTRACT Lyophilized aryl phosphate monoesters having a pH of substantially l to 4 upon reconstitution with the amount of water removed during lyophilization; are prepared by lyophilizing an aqueous solution of the aryl phosphate monoesteis having a pH of substantially l to 4. Diagnostic compositions for the determination of phosphates in which the lyophilized aryl phosphate monoesters are employed.

7 Claims, No Drawings LYOPHILIZED ARYL PHOSPHATE MONOESTERS AND PROCESS THEREFOR The present invention'relates to novel lyophilized aryl phosphate monoesters, more particularly phenyl phosphate or p-nitrophenyl phosphate, which have a pH of substantially l to 4 upon reconstitution with that volume of water removed during lyophilization; the process for their preparation; and diagnostic compositions in which such a lyophilized monoester can be usefully employed for the determination of phosphatase.

Though phenyl phosphate and pnitrophenyl phosphate can be employed for various chemical purposes, for example as chemical intermediates or as buffers, their widest use is in biochemistry as substrates in assay procedures designed to determine phosphatase, i.e.; phosphomonoesterase, levels in various body .fluids. It is known, for example, that alteration of phosphate. The hydrolysis reaction has been suggested to be depicted as follows:

RO-P-OH (free acid) P03- ROH I, RO--P0- Rgi 0- (monoionic) (diionic) Phosphatase is considered to catalyze the final step of the above hydrolysis. The term p-nitrophenyl phosphate and phenyl phosphate are defined in this application to signify the free acid form, the monoionic form, and the diionic form represented by the forego-- ing general structures. The source of the p-nitrophenyl phosphate and phenyl phosphate is either the free acid, ROH PO or an ammonium, alkali metal or alkaline earth metal salt thereof. in this application, the terms p-nitrophenyl dihydrogen phosphate and phenyl dihydrogen phosphate specifically indicate the free acid which for the purposes of this invention is equivalent to the ammonium, alkali metal or alkaline earth metal salts thereof.

In clinical chemistry, phosphatases are referred to as acid" or alkaline, the terms indicating the optimum pH at which they are measured. The usual procedure for the determination of either acid or alkaline phosphatase involves the incubation of a phosphate monoester such as p-nitrophenyl phosphate or phenyl phosphate in an appropriately buffered medium with a sample of the fluid to be analyzed for the presence of phosphatase. The above depicted hydrolysis reaction is allowed to occur and thereafter the mixture then is 'analyzed either for inorganic phosphate or the presence of free organic alcohol, ROH, which comprised the organic portion of the phosphate monoester,

to'provide an indication of the levels of phosphatase present. Because the rate of hydrolysis is actually the determinative factor in the phosphatase assay, it is particularly important that the reactants be substantially unhydrolyzed prior to use in the assay. In the case of materials known to be prone to absorption of moisture, it is particularly important that such materials be applied in an anhydrous form in order to avoid the inhibition of the enzyme activity attributable to the partial hydrolysis accompanying such hydration. Though pnitrophenyl dihydrogen phosphate and phenyl dihydrogen phosphate are reputed to be particularly convenient substrates for use in phosphatase determination, their proclivity for hydration and consequent hydrolysis limits their usefulness. Consequently it was desired to prepare a phenyl dihydrogen phosphate and a p-nitrophenyl dihydrogen phosphate which would be resistant to hydrolysis and able to endure longer shelf life prior to use in a phosphatase assay.

It has been surprisingly found that preparing a solution of phenyl phosphate or p-nitrophenyl phosphate which has a pH of substantially l to 4 and most preferably a pH of substantially l, and subjecting that solution to lyophilization enhances the resistance to hydrolysis of the resultant lyophilized material. Such a preparation may be suitably achieved by mixing the components of phenyl dihydrogen phosphate or pnitrophenyl dihydrogen phosphate or the alkali metal, ammonium or alkaline earth metal salts thereof in such porportions as to form a solution having a pH within the desired range of substantially l to 4 and preferably about pH 1. YA solution of such pH range can also be prepared by dissolving a quantity of phenyl dihydrogen phosphate or p-nitrophenyl dihydrogen phosphate or an alkali metal, ammonium or alkaline earth metal salt thereof in water and adjusting the pH of the resultant solution. Most preferably, a solution of desired pH can be obtained by acidifying a quantity of water to a pH within the desired range and adding to that acidified solution a quantity of phenyl dihydrogen phosphate, pnitrophenyl dihydrogen phosphate or an alkali metal, ammonium or alkaline earth metal salt thereof sufficient to dissolve in the acidic aqueous media without significantly changing the pH from the desired level. Any acid of sufficient strength may be used in the adjustrnent of the pH except that when the end product is destined for use in a phosphatase assay, acids which contribute anions which may interfere with enzymatic activity such as phosphoric acid or sulfuric acid should not be employed. The contemplated lyophilization can be desirably carried out in a freeze dryer (lyophilizer) at shelf temperatures of about 20 to 30 C. for a period of 48 to 96 hours and most preferably at a temperature of about 25 for a period of about 48 to 72 hours. Suitable ranges of pressure are approximately to 25 microns while most preferably the pressure is maintained at about 25 microns. It will be apparent to one skilled in the art that lyophilization temperatures and pressures are interdependent, lower temperatures resulting in higher pressures. Of course, the lyophilization can be carried out at higher or lower temperatures with a commensurate loss in stability as a result of the tendency towards hydrolysis at higher temperatures and moistures, and extension of time of drying at lower temperatures. Most desirably, it has been found that the moisture of the matrix should be maintained at about 3 percent since this provides good stability.

The pH of the solution to be lyophilized is critical. As previously specified this pH must be substantially 1 to 4, with the most preferred pH being substantially l. The term substantially is used to indicate that the pH adjustment can be made to a point a few tenths of a pH point below 0.5 or above 4 without unacceptably altering the amount of hydrolysis during and for extended periods after lyophilization. Thus when the lyophilization was carried out at pH 5 an unacceptable change in absorbence at about 400 millimicrons of from about 0.09 to about 0.4, after storing the lyophilized material for 22 hours at 40 C., was observed. An unhydrolyzed material would show substantially no absorbence, ie

less than 0.1, upon-reconstitution with about 400 times the volume of water in the mixture prior to lyophilization. At a lyophilization. pH of 8, absorbence immediately after lyophilization was an unacceptable 0.3

and after 22 hours at 40 C. was an even more unacceptable 0.94. At a lyophilization pH of 12, absorbence immediately after lyophilization was an unacceptable 0.74 and after 22 hours at 40 C. was more than 1.5.

The resistance to hydrolysis exhibited by the lyophilized product of the instant invention is particularly unexpected in view of the known fact that aqueous solutions of alkyl and aryl phosphate monoesters exhibit minimum or essentially no hydrolysis at a pH of around 7 or higher while greatest hydrolysis is observed at between pHs of almost to 6, with maximum velocity at about pH 4. However, we have found a completely opposite effect where frozen solids and lyophilization are concerned.

The lyophilized product obtained accordingto the procedure outlined above has the added advantage of being in the dry state which permits the material to be packaged in containers which are easy to handle, store e.g. at C. preferably in the absence of light, and use. Further, each of the containers may include a quantity of the material which is exactly that amount required for making a particular number of phosphatase determinations or for a single phosphatase determination. in order to make one such phosphatase determination, the contents of one standard size container may be mixed with a predetermined quantity of aqueous buffer to produce a liquid reagent that is suitable for making a single assay. For the determination of phosphatase activity, it is necessary to use an acidic or alkaline buffer dependent upon whether acid or alkaline phosphatase is to be determined. The buffer selected should be characterized by a pH and pK of about the pH needed for maximum velocity of the enzyme reaction which is being assayed. An example of a suitable buffer for alkaline phosphatase is 2-amino-2-methyl-l-propanol as illustrated in Example 6 following while a suitable buffer for acid phosphatase is illustrated by the citrate buffer exemplified in Example 7 following. Alternatively, the lyophilized product of this invention may be formed into tablets, pills or other forms which can provide diagnostic compositions which are usefully employed in the determination of phosphatase. Additionally, the diagnostic compositions of this invention can be adherent in an inert solid support material. For

example, said compositions may be applied to splinters, sticks or strips made of wood fiber including paper, glass, metal or plastic. Particularly preferred are bibilous materials such as paper, wood fiber or the like having any desired shape which readily absorb a solution of the lyophilized material of this invention to form an impregnated bibilous mat of paper. Alternatively, it has been discovered that such bibilous strips may be impregnated with a solution of p-nitrophenyl phosphate or phenyl phosphate which has been adjusted or prepared such that it exhibits a pH of substantially l to 4 without the need for lyophilization of this solution followed by reconstitution. Drying of the strip which has been impregnated with the substantially pH 1 to 4 solution provides a ready form of diagnostic composition which is characterized by enhanced stability, longer shelf life and convenient form for simple and accurate usage by the clinician.

As indicated above, the p-nitrophenyl dihydrogen phosphate and phenyl dihydrogen phosphate can be used in their free acid form as starting materials for the lyophilization process, but the ammonium, alkali metal and alkaline earth metal salts thereof are preferred from the standpoint of commercial availability. Moreover, the alkali metal salts such as lithium, potassium and especially sodium are particularly convenient for use. A most preferred embodiment of this invention is a lyophilized p-nitrophenyl phosphate which has been derived from disodium p-nitrophenyl phosphate by dissolution of that material in acidic aqueous media having a pH of substantially 1 followed by subjection to lyophilization. The product thus obtained upon reconstitution with the amount of water removed during lyophilization exhibits a pH of substantially 1.

The free organic phosphate monoesters of this invention can be obtained by any standard procedure, such as the passage of an alkali metal salt through a cationic exchange resin, e.g., Dowex 50 inthe acid form. The ammonium, alkali metal and alkaline earth metal salts can be obtained by the addition of an excess of the appropriate hydroxide to a solution of the free organic phosphate monoester, followed by evaporation of the solvent or by ion exchange using for example as a starting material the readily available sodium salt.

The following examples will further illustrate specific embodiments of the invention. These examples are set forth by way of illustration only and it will be understood that the invention is not to be construed as limited either in spirit or in scope by the details contained therein as many modifications and substitutions in both materials and methods will be apparent from this disclosure to those skilled in the art. Percentages refer to percent by weight unless otherwise indicated, relative amounts of materials are expressed in parts by weight, except as noted otherwise, and temperatures are given in C.. The relationship between parts by weight and parts by volume is the. same as that existing between grams and milliliters. The abbreviations used are as follows: milliliters: ml. Molar: M; millimicrons: m;:.. for wavelength oflight; Normality: N.

EXAMPLE 1 Parts by volume of a pH 10.43 solution containing 9.075 parts of disodium p-nitrophenyl phosphate in deionized water was divided into 20 aliquots containing 5 parts each. To six of these aliquots was added sufficient hydrochloric acid to attain pH levels of 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 respectively, as determined by a 'pH meter. After pH adjustment, the solution was placed in a freeze-dryer (lyophilizer) on shelves having' a temperature of about -40 to about 5 C. After the liquid was frozen, vacuum was applied to the chamber until the pressure reached about 200 microns. At that point, the condenser was cooled and the shelves were heated to about 20 C (at this shelf temperature, the pressure in the chamber was about 50 microns and the temperature of the condenser was about 50 C). The vials were lyophilized for 72-96 hours under the above conditions. At the end of that time nitrogen was allowed to enter the chamber until inches of vacuum was reached. The vials were then stoppered in the freeze-dryer with the stoppering plate provided therein. After removal from the freeze-dryer, the vials are further sealed by crimping aluminum seals over the stoppers. Upon reconstitution of the lyophilized material present in each of the vials prepared above, no

appreciable change in pH was observed to have oc.

curred during lyophilization.

EX AM PLE 2 About 20 parts of water was adjusted to a pH of 1.1 i 0.05 with aqueous hydrochloric acid. To this solution was added 0.32 part of disodium p-nitrophenyl phosphate. The resulting solution was determined to have a pH of 1.4. One part by volume of this solution waspipetted into a vial and subjected to the lyophilization procedure described in the second paragraph of Example 1 except that shelf temperatures of about 20-30 C. and a lyophilization period of about 48-72 hours were employed rather than those described in Example 1. The resultant lyophilized material showed no appreciable absorbance at about 400 millimicrons when reconstituted with water.

EXAMPLE 3 100 Parts by volume of a solution containing 2 parts of dipotassium phenyl phosphate in deionized water was divided into 6 aliquots. To each of these aliquots was added sufficient hydrochloric acid to achieve pH levels of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 respectively, as determined by a pH meter. After pH adjustment, the vials were treated identically with'those prepared according to Example 1 and thereafter lyophilized according to the procedure described in the second paragraph of Example 1 to provide a stable lyophilized material.

EXAMPLE 4 An equivalent quantity of dimagnesium p-nitrophenyl phosphate, was substituted for the disodium pnitrophenyl phosphate in Example 2 and the procedures described therein are repeated. The lyophilized material thus obtained shows no appreciable change in absorbance at about 400 millimicrons when reconstituted with water.

EXAMPLE 5 An equivalent quantity of diammonium p-nitrophenyl phosphate, was substituted for the disodium pnitrophenyl phosphate in Example 2 and the procedures described therein are repeated.

' EXAMPLE 6 Diagnostic Composition For The Determination of Alkaline Phosphatase.

l. Buffer: milliliters of 0.84 M 2-amino-2- methyl-l-propanol buffer having a pH of about 10.2.

2. Phosphate Substrate: Vialseach containing 0.84 micromoles of the lyophilized disodium p-nitrophenylphosphate prepared according to Example 2 and 0.0105 micromoles of magnesium chloride are stored at refrigerated temperatures.

21 Ml. of the buffer was added to a vial containing v the phosphate substrate, with mixing. This working reagent is stable for at least 2 months when refrigerated.

0.5 Ml. of this solution was added to each of two cuvettes, and the cuvettes placed in a water bath at 37 C. for 5 minutes. To one of the cuvettes was added with mixing 0.02 ml. of the specimen to be tested for phosphatase, e.g., serum or plasma; this cuvette serving as a test sample. The other cuvette to which no specimen was added at that time, served as a blank. The cuvettes were incubated at 37 C. for exactly 30 minutes. At the end of the incubation period, 5 ml. of 0.05 N sodium hydroxide, was added with mixing to each of the cuvettes. To the blank cuvette there was then added 0.02 ml. of the specimen.

Standards were prepared using a known concentration of p-nitrophenol, suitably 10 ml. of 0.36 micromoles.

The'intensity of the color developed in the. test samples and blanks was read at any of the wave lengths between 400 to 420 millimicrons in a colorimeter or spectrophotomer in which the absorbance of a water blank was set at zero. Standards are read at the same wave length used to read the test samples and the blanks. The actual absorbance of the test sample is calculated by subtracting the blank absorbance from the sample absorbance. The actual absorbance is compared with the reading obtained with the reference standard to estimate the amount of phosphatase in the specimen.

EXAMPLE 7 Preparation of diagnostic composition for the determination of acid phosphatase.

1. Buffer: A 0.22 M citrate buffer, of pH 4.8 is prepared by dissolving 42 grams of citric acid in 400 ml. of 1 N aqueous sodium hydroxide, then diluting the solution to 1000 ml. with distilled water. i

2. Phosphate substrate: 3 grams of the lyophilized disodium phenyl phosphate prepared according to Example 3 is dissolved in 250 milliliters of buffer prepared in the foregoing paragraph.

The test employing this diagnostic composition may be carried out in a manner conventional for acid strate, 1 milliliter of the citrate buffer and 0.2 milliliter of the specimen to be analyzed for acid phosphatase is incubated at 37 C. for exactly 5 minutes. At the end of the incubation period, the reaction is stopped and the mixture made alkaline by the addition of 0.375 N aqueous sodium hydroxide. To this mixture there is then added 1 milliliter, of buffered aminoantipyrine solution (This solution can be prepared by mixing grams of 4-aminoa'ntipyrine, 30 grams of sodiumcarbonate and 30 grams of sodium bicarbonate, then diluting with distilled water to a final volume of 1000 milliliters.) with mixing, followed .by the addition of 1 milliliter of buffered potassium ferricyanide solution (This solution is prepared by diluting a mixture of 40 grams of potassium ferricyanide, grams of sodium carbonate and grams of sodium bicarbonate to 1000 milliliters with distilled water.) to result in the formation of a red solution the absorbance of which is measured at 505 millimicrons mu. A control sample identical to the incubation mixture except that-no specimen ispresent is also subject to the above procedure, thus serving as the reagent.

The amount of phenol released during the incubation can be estimated with reference to working standards containing a known concentration of phenol and thus the activity of the acid phosphatase in the serum can be determined. Likewise, a control serum of known'acid phosphatase, activity can be used as a reference standard.

What is claimed is:

l. A lyophilized monophenyl phosphate or mono-pnitrophenyl phosphate having a pH'of substantially l to 4 upon reconstitution with that volume of water removed during lyophilization of an aqueous solution of the respective monoester or the ammonium, alkali metal or alkaline earth metal salts of said monoester, which solution is acidified to a pH of substantially 1 to 2. The composition of claim 1; wherein the phosphate is a mono p-nitrophenyl phosphate.

3. The composition of claim 2, wherein the phosphate has a pH of substantially 1, upon reconstitution with that volume of water removed during lyophilization.

4. The process for the preparation of lyophilized mono-phenyl dihydrogen phosphate or mono-pnitrophenyl dihydrogen phosphate and the ammonium alkali metal and alkaline earth metal salts thereof, which comprises lyophilizing an aqueous solution of said monoester, such aqueous solution having been acidified to a pH of substantially l to 4.

5. The process according to claim 4, where the lyo philization is carried out at a temperature of from about 20 to 30 C. and a pressure of about to 25 microns. 4

6. According to claim 4, the process wherein the monoester is mono-p-nitrophenyl dihydrogen phosphate or an alkali metal salt thereof and the pH is substantially l.

7. According to claim 6, the process wherein said monoester is disodium mono-p-nitrophenyl phosphate. 

2. The composition of claim 1, wherein the phosphate is a mono p-nitrophenyl phosphate.
 3. The composition of claim 2, wherein the phosphate has a pH of substantially 1, upon reconstitution with that volume of water removed during lyophilization.
 4. The process for the preparation of lyophilized mono-phenyl dihydrogen phosphate or mono-p-nitrophenyl dihydrogen phosphate and the ammonium alkali metal and alkaline earth metal salts thereof, which comprises lyophilizing an aqueous solution of said monoester, such aqueous solution having been acidified to a pH of substantially 1 to
 4. 5. The process according to claim 4, where the lyophilization is carried out at A temperature of from about 20* to 30* C. and a pressure of about 70 to 25 microns.
 6. According to claim 4, the process wherein the monoester is mono-p-nitrophenyl dihydrogen phosphate or an alkali metal salt thereof and the pH is substantially
 1. 7. According to claim 6, the process wherein said monoester is disodium mono-p-nitrophenyl phosphate. 